The conversion of lactose to GOS by
The conversion of lactose to GOS by β-gal involves the same transglycosylation mechanism used by most glycoside hydrolases (GHs). This mechanism (Fig. 1) involves an enzyme-bound intermediate and consists of two processes: glycosylation and deglycosylation (Adlercreutz, 2017). The glycosylation process results in the release of glucose and the formation of an enzyme-galactose intermediate through cleavage of the β-1,4 glycosidic bond of lactose by a nucleophilic amino acid. Glycosylation is catalyzed by another amino vuf acting as a general acid-base. The deglycosylation process produces GOS through transfer of the covalently attached galactose molecule to another molecule of lactose (or other sugar). In a competing process called hydrolysis, the enzyme-galactose intermediate can transfer the covalently attached galactose to a water molecule and produce galactose (Brás, Fernandes, & Ramos, 2010). During deglycosylation, water and lactose (or other sugar) are in a competitive relationship. To increase GOS yields, the competitiveness of lactose in the deglycosylation process must be enhanced. The reaction mechanism presents three strategies to improve transglycosylation by GHs (Pontus Lundemo, Karlsson, & Adlercreutz, 2017). The first strategy involves modification of the enzyme’s aglycone(+) site to increase the enzyme’s affinity for the substrate (Armand et al., 2001, Taira et al., 2010). For example, Johansson et al. (Johansson et al., 2004) reported that an aromatic amino acid in the aglycone site is important for the interaction of enzymes in GH family 16 with glycosides. The second strategy involves modifying the enzyme’s glycone(−) site to lower its affinity for the glycone(−) (Arab-Jaziri et al., 2015, Jr et al., 2006). For example, Teze et al. (2014) mutated a highly conserved sequence around the −1 site of Thermus thermophilus β-glycosidase to obtain mutants with improved synthesis capability. The third strategy involves increasing the hydrophobicity of the enzyme’s active site to decrease its affinity for competing water molecules (Frutuoso and Marana, 2013, Honda et al., 2008, Lundemo et al., 2013). For example, Honda et al. (2008) reported that mutating a residue holding the nucleophilic water molecule with the general acid/base residue can convert an inverting enzyme to a glycosynthase.
In this study, we performed a structural and functional analysis of Aoβ-gal that allowed us to rationally design Aoβ-gal variants exhibiting improved (by up to 59.8%) GOS yields. Two different strategies to improve transglycosylation efficiency were studied. The first strategy was to change a conserved residue at the −1 site to destroy the hydrogen bond between the enzyme and the aglycone. The second method was to increase the hydrophobicity around the active center. When combined, the two strategies increased the GOS yield to a higher degree, offering a new way to improve the transglycosylation efficiency of a GH family member.
Materials and methods
Results and discussion
Conclusion Two single mutants (N140C and W806F) and one double mutant (N140C/W806F) of Aoβ-gal, were constructed, and their GOS yield from lactose was determined. Using optimal temperature and pH values for each enzyme variant, we found that N140C, W806F and N140C/W806F all produced GOS yields higher than that of Aoβ-gal. N140C/W806F produced the highest GOS yield reported to date, which addresses the bottleneck associated with Aoβ-gal’s low GOS yield and significantly improves its potential industrial application. The mechanism underlying the increased transglycosylation activity of N140C and N140A involved the disruption of galactose binding, which decreased the likelihood of water acting as the acceptor substrate and favored the use of a sugar as the acceptor substrate. W806F increased active-site hydrophobicity to enhance transglycosylation. The activity of N140C/W806F reflects an additive effect of the two single mutants to further improve the transglycosylation activity of Aoβ-gal. This study introduces an improved variant of Aoβ-gal that is suitable for industrial applications and provides an important theoretical basis for further research into the mechanisms of β-gal reactions.